Activation Of Glu-plg Research Articles

Mechanism of the synergistic effect between oversulfated chondroitin-6-sulfate and lysine or 6-aminohexanoic acid in enhancing the in-vitro activation of glutamic plasminogen by tissue plasminogen activator or urokinase

Earlier studies of addition of naturally sulfated polysaccharides including unfractionated heparin showed a significant enhancement of the in-vitro activation of glutamic plasminogen (Glu-Plg) by tissue plasminogen activator (t-PA) or urokinase plasminogen activator (u-PA). However, supplementing of physiological concentration of NaCl (0.9%) to the buffer reversed the enhancement. To overcome this reversal attempts were made to oversulfate the compounds and re-evaluate their biological properties. Chondroitin-6-sulfate (N-2) was oversulfated using chlorosulfonic acid-pyridine complex and isolated as the sodium salt. Infrared and H-NMR studies of the oversulfated compound showed introduction of new sulfate groups with the formation of 60% of chondroitin-4-6-disulfate. In-vitro studies were conducted on comparing the effect of oversulfated chondroitin-6-sulfate (S-2) with native compound (N-2) and unfractionated heparin in enhancing the activation of Glu-Plg by t-PA or u-PA using 0.05 mol/l Tris buffer (pH 7.3) containing 0.9% of NaCl. The enhancement of activation of Glu-Plg by t-PA or u-PA was measured by formation of plasmin using H-D-Glu-Phe-Lys-pNA (S-2403) as the substrate. The activation by t-PA was enhanced two-fold by 2.86 microg/ml of S-2, 4-6-fold by addition of 32.4 mmol/l of lysine or 5.4 mmol/l of 6-aminohexanoic acid (6-AH) and 14-16-fold enhancement by addition of both S-2 and lysine or S-2 and 6-AH showing a synergistic effect, whereas unfractionated heparin alone gave no enhancement and in conjunction with lysine or 6-AH gave no additional enhancement. Similar studies using u-PA in place of t-PA gave identical results. During the activation of Glu-Plg to plasmin, lysine plasminogen (Lys-Plg) is reported to be an intermediate. Therefore we investigated the role of S-2, lysine and 6-AH in the activation of Lys-Plg to plasmin. The results showed that S-2 enhanced this activation, whereas lysine or 6-AH which were active in enhancing the activation of Glu-Plg were not active using Lys-Plg indicating that the site of enhancement by lysine or 6-AH was during the initial phase. Double reciprocal plot of Glu-Plg activation by t-PA with or without S-2 and lysine showed no change in Km but a 10-fold increase of Kcat suggesting a template mechanism for the attenuation when both cofactors are used.

Effect of native fucoidan, sulfated fucoidan, heparin and 6-aminohexanoic acid on the activation of glutamic-plasminogen by urokinase: role of NaCl

Addition of a physiological concentration of NaCl (0.9%) to 0.05 mol/l Tris buffer (pH 7.4) reversed the enhancement of the activation of glutamic-type plasminogen (Glu-Plg) by low molecular weight urokinase by fucoidan and heparin, while addition of aminohexanoic acid (6-AH) enhanced the activation. Native fucoidan (N-2), sulfated fucoidan (S-2) and heparin alone and in the presence of 6-AH were investigated to determine the effect of NaCl addition to 0.05 mol/l Tris (pH 7.4) on the activation of Glu-plg by high molecular weight urokinase (HMW u-Pa). Heparin alone and in conjunction with 6-AH enhanced 4.5 to 5.5-fold the initial rate of activation of Glu-plg, while N-2 alone or in conjunction with 6-AH gave 3 to 5.5-fold enhancement and S-2 gave no enhancement of activation using 0.05 mol/l Tris buffer (pH 7.4). Addition of 0.9% NaCl to the buffer reversed the enhancement by the cofactors but, in the presence of 6-AH, N-2 gave two-fold and S-2 gave three-fold enhancement of activation while heparin gave 25% enhancement. The mechanism of enhancement by S-2 in the presence of 6-AH was investigated by dilution and enzyme kinetic studies. The results show that the enhancement was due to interaction with HMW u-PA and not with Glu-plg.

Mechanism of the stimulatory effect of native fucoidan, highly sulfated fucoidan and heparin on plasminogen activation by tissue plasminogen activator: The role of chloride

Native Fucoidan and unfractionated heparin enhanced by 6-fold the in vitro activation of human glutamic plasminogen (Glu-Plg) by tissue plasminogen activator (t-PA) using 0.05M Tris buffer pH 7.4, while sulfated fucoidan inhibited the activation under these conditions. Double reciprocal plots of these interactions showed that sulfated fucoidan inhibited the activation in a noncompetitive manner while the enhancements by heparin or native fucoidan were due to an increase of Vmax without affecting Km. To determine whether the stimulatory effect of the individual cofactor was due to its interaction with Glu-Plg or with t-PA, experiments were performed at a fixed level of the cofactor and either varying in a serial fashion the concentration of Glu-Plg or of t-PA. The ratios of the initial rate of plasmin generation in the presence or absence of the cofactors were plotted against the inverse of the volume fraction of Glu-Plg or of t-PA. The results showed that heparin interacted with Glu-Plg while native fucoidan and sulfated fucoidan interacted with t-PA. Studies were also conducted on the effect of the two fucoidans and heparin on the activation of Glu-Plg by t-PA using 0.05M Tris buffer pH 7.4 containing 0.1 M NaCl. Under these conditions, sulfated fucoidan was most effective in enhancing the activation followed by native fucoidan and heparin respectively. The results of this study showed that in presence of the buffer containing 0.1 M NaCl, heparin was interacting with t-PA while the two fucoidans were interacting with both t-PA and Glu-Plg. A comparison of the double reciprocal plots of the rate of enhancement by the cofactors using 0.05M Tris buffer pH 7.4 containing 0.1M NaCl or in presence of buffer alone showed that the cofactors were more effective using 0.05M Tris buffer pH 7.4 alone and that addition of NaCl to the buffer slowed down the reactions by decreasing Vmax without affecting Km. Circular Dichroism (CD) studies of Glu-Plg in the far ultraviolet (UV) range showed that addition of NaCl destabilized the beta sheet structure which was reversed by addition of 6-aminohexanoic acid (6-AH) or one of the cofactors, while the near UV CD spectra of Glu-Plg in presence of 0.1 M NaCl was enhanced by the cofactors by increasing its ellipticity as reported earlier for 6-AH.

Mechanism of the stimulatory effect of 6-aminohexanoic acid on plasminogen activation by streptokinase or tissue plasminogen activator: the role of chloride.

Studies were conducted on the mechanism of the stimulatory effect of 6-aminohexanoic acid (6-AH) during the in vitro activation of human glutamic plasminogen (Glu-Plg) by streptokinase or by tissue plasminogen activator (t-PA) and the possible role of the addition of physiological concentrations of NaCl to the buffer solution. Enhancement by 6-AH was investigated by measuring the rate of plasmin generation using chromogenic substrate H-D-glu-phe-lys-pNA (S-2403). Control studies using plasmin showed that the addition of 6-AH at concentrations below 20 mM did not significantly affect the initial rate of the amidolytic activity of plasmin with or without the addition of NaCl to 0.05 M Tris buffer (pH 7.4). On the other hand, addition of NaCl to the buffer slowed down the initial rate of activation of Glu-Plg by streptokinase or by t-PA while increasing the percent enhancement by 6-AH when compared with the controls. The ratios of the initial rates of plasmin generation in the presence or in the absence of 6-AH were plotted against the inverse of the volume fraction of Glu-Plg, streptokinase or t-PA after serial dilutions. The results showed that when the activation reactions were performed in 50 mM of Tris buffer (pH 7.4), the enhancements by 6-AH were related to its interaction with streptokinase or t-PA, while using the same Tris buffer containing 0.6 % NaCl, the enhancements by 6-AH were related to its interaction with both Glu-Plg and streptokinase or t-PA. However, upon increasing the NaCl to 0.9%, the results showed that the enhancements by 6-AH of the activation of Glu-Plg by streptokinase or t-PA were related to its interaction with Glu-Plg. The results suggested that changes in the concentrations of NaCl play a regulatory role during the activation process.

Ionic modulation of the effects of heparin and 6-aminohexanoic acid on plasminogen activation by streptokinase: The role of ionic strength, divalent cations and chloride

Studies were conducted on the effect of heparin or 6-aminohexanoic acid (6-AH) on the activation of glutamic plasminogen (Glu-Plg) by streptokinase in the presence of different concentrations of buffer, NaCl and divalent cations. Heparin and 6-AH inhibited streptokinase-mediated activation of Glu-Plg using 10 mM Tris-HCl buffer pH 7.4. This inhibition was partially reversed by the addition of 0.2-1.0 mM of Mg ions. Increasing the ionic strength of Tris-HCl buffer from 10 to 50 mM or addition of 50-150 mM of NaCl to 50 mM Tris-HCl pH 7.4 inhibited the activation of Glu-Plg by streptokinase while decreasing the % inhibition by heparin over the control samples. Double reciprocal plot of the activation of Glu-Plg by streptokinase using 50 mM Tris-HCl pH 7.4 containing 100 mM NaCl showed that the addition of heparin lowered Vmax by 50% without affecting Km. To determine whether the inhibitory effect of heparin was specifically directed towards Glu-Plg or streptokinase, the ratios of the initial rate of plasmin generation in the presence of heparin over the controls were plotted against the inverse of the volume fraction of Glu-Plg or streptokinase after serial dilutions. The results indicated that the dilutions of streptokinase but not of Glu-Plg influenced the ratios, suggesting an interaction of heparin with streptokinase. Addition of 6-AH reversed the inhibitory effect of NaCl on the activation of Glu-Plg by streptokinase and the results of the near UV CD spectra of Glu-Plg showed that addition of 6-AH enhanced the spectra in this region with an increase in the ellipticity which was not affected by addition of NaCl.

The effect of 6-aminohexanoic acid and fucoidan on the activation of glutamic plasminogen by streptokinase.

Studies were conducted on the effect of 6-aminohexanoic acid (6-AH) or fucoidan on the activation of glutamic plasminogen (glu-plg) by streptokinase using 0.05 mol/l Tris buffer containing a physiological concentration of NaCl. In contrast to the earlier reports where no NaCl was added to the buffer solution, addition of 6-AH enhanced the initial rate while the inhibition by fucoidan was not affected. Double reciprocal plots of the activation of glu-plg by streptokinase in the presence of 6-AH showed an increase in Vmax, but no change in Km. However, the addition of fucoidan showed a decrease in Vmax, but no change in Km. To determine whether the stimulatory effect of 6-AH was specifically directed towards glu-plg or streptokinase, the ratios of the initial rate of plasmin generation in the presence of 6-AH over the controls were plotted against the inverse of the volume fraction of glu-plg or streptokinase after serial dilutions. The results indicated that the dilutions of glu-plg, but not of streptokinase, influenced the ratios, suggesting an interaction of 6-AH with glu-plg. Similar experiments were conducted to determine the mechanism of inhibition of streptokinase by fucoidan. The results indicated that fucoidan was interacting with streptokinase, but not with glu-plg. Circular dichroism studies of glu-plg in the near-ultraviolet spectra (250-308 nm) showed that addition of 6-AH enhanced the spectra in the region around certain chromophores, which reflected conformational changes. On the contrary, the far-ultraviolet spectra were almost identical.

Effect of fucoidan during activation of human plasminogen

Fucoidan [sulfated poly (L-fucopyranose)] was compared with 6-aminohexanoic acid (6-AH) or CNBr-cleaved fibrinogen (CNBr-Fbg) alone or in combination in enhancing the activation of glutamic plasminogen (Glu-Plg) or lysine plasminogen (Lys-Plg) by two-chain tissue plasminogen activator (t-PA) or LMwt-urokinase or by streptokinase. Fucoidan enhanced the t-PA activation of Glu-Plg or Lys-Plg at Plg concentrations greater than 75nM, while stimulation by CNBr-Fbg of t-PA activation followed saturation kinetics of Michaelis-Menton. During t-PA activation of Glu-Plg, a high degree of synergism was observed between 6-AH and fucoidan while the enhancement by CNBr-Fbg was not influenced by fucoidan and was reversed by 6-AH. Fucoidan alone at higher concentrations was effective in enhancing the activation of Glu-Plg by urokinase while the combination of fucoidan and 6-AH showed additive effect in enhancing the activation of Lys-Plg. The activation of Glu-Plg by streptokinase was reversed by fucoidan in a manner similar to that reported for 6-AH. The results are interpreted to suggest that CNBr-Fbg and 6-AH compete with each other for the same lysine binding sites (LBS) on the Plg molecule while fucoidan acted synergistically with 6-AH in enhancing the t-PA activation of Glu-Plg by a different mechanism. The double reciprocal plot for the interaction of Glu-Plg and urokinase also showed a significantly higher affinity between the two in presence of fucoidan.

Effects of N-terminal peptide of glu-plasminogen on the activation of glu-plasminogen and its conversion to lys-plasminogen

In order to analyze the mechanism of the intramolecular binding of the N-terminal peptide of Glu-plasminogen (Glu-plg) to its kringles, which results in its tight conformation, we synthesized peptides of the N-terminal portion of Glu-plg molecules and analyzed their effects on the activation of Glu-plg and its conversion to Lysplasminogen (Lys-plg) by plasmin. Three peptides of Ala 44-Lys 50, Ala 44-Glu 51 and Ala 44-Ser 49 were synthesized in order to examine the effect of lysine residue in the peptide. Ala 44-Lys 50 and Ala 44-Glu 51 enhanced the activation of Glu-plg by urokinase, whereas the activation of Lys-plasminogen (Lys-plg) was slightly inhibited. The conversion of Glu-plg to Lys-plg by plasmin was also enhanced by these peptides. The results suggest that Ala 44-Lys 50 and Ala 44-Glu 51 worked on Glu-plg in a similar manner as lysine analogues by making its conformation looser. The third peptide Ala 44-Ser 49 did not have any effect on the activation of Glu-plg by urokinase or the conversion of Glu-plg to Lys-plg by plasmin. Ala 44-Lys 50 residue of Glu-plg is, therefore, strongly implicated as a candidate for the responsible site of the intramolecular binding in Glu-plg.

Enhancement of the activation of Glu-plasminogen by urokinase in the simultaneous presence of tranexamic acid or fibrin.

The activation rate of Glu-plasminogen (Glu-plg) by urokinase (UK) was enhanced in the presence of either tranexamic acid or fibrin with an increase in the catalytic rate constant (kcat). The maximum increase in kcat was obtained at 0.5 mM of tranexamic acid and 0.1 microM of fibrin. Km did not change. The addition of fibrin to 1 mM tranexamic acid resulted in a further increase in kcat of the UK activation of Glu-plg. On the other hand, the addition of tranexamic acid to 0.1 microM fibrin further increased kcat of the UK activation of Glu-plg. Thus, stimulatory effects were observed on the activation of Glu-plg by UK in the simultaneous presence of tranexamic acid and fibrin. Fibrin-binding sites on the kringle 5 of Glu-plg may be involved in the further increase in the activation rate of Glu-plg by UK in the presence of both fibrin and tranexamic acid in comparison to that in the presence of tranexamic acid alone. Possibly, the Glu-plg binding with both tranexamic acid and fibrin (at kringle 5) may be most effectively activated by UK. It is also suggested that two molecules of Glu-plg bind to one molecule of fibrin monomer.

Differences in the activation rates of plasminogen by tissue plasminogen activator and urokinase

The activation of a native form of plasminogen (Glu-plg) by tissue plasminogen activator(t-PA) was enhanced when the plasma was clotted by the addition of thrombin or thrombin plus Ca ++. Cross-linking of fibrin in the clotted plasma did not inhibit the fibrin-associated enhancement of the activation of plasminogen by t-PA. When fibrinolysis induced by t-PA in the clotted plasma was measured using enzyme immunoassay, lysis of non cross-linked fibrin in the clotted plasma was faster than lysis of cross-linked fibrin, however such decrease in the extent of fibrinolysis was observed in cross-linked fibrin even in the absence of α 2mantiplasmin (αAP) in a purified system. When Glu- or Lys-plg (modified plg) was activated by t-PA, the presence of fibrin enhanced significantly the extent of activation of both Glu- and Lys-plg, but the activation of Glu-plg by urokinase (UK) was enhanced in the presence of fibrin. The activation of Lysplg by UK was rather inhibited in the presence of fibrin.

フィブリン，フィブリノーゲンよりのＢβペプチドの遊離 血しょう中と純化した系での比較

Release of Bβ peptides and F (g) DP from fibrin (ogen) was studied after the activation of Glu-plasminogen (Glu-plg) by urokinase (UK) in the plasma or clot and fibrinogen or fibrin. Bβ peptides or FDP were released faster from the clot than the plasma. In a purified system, FDP was released faster than FgDP after the activation of Glu-plg by UK in the presence of fibrin or fibrinogen. Release of Bβ 15-42 from purified fibrin was slower than the release of Bβ 1-42 from fibrinogen when Glu-plg was activated by UK. The presence of α2 antiplasmin (α2AP) slowed the release of Bβ 1-42 from fibrinogen, thus resulted in faster release of Bβ 15-42 in comparison to Bβ 1-42. These results indicated that Glu-plg was activated better by UK in the presence of fibrin than fibrinogen, but the release of Bβ peptides from purified fibrin and fibrinogen depended upon the presence of α2AP. Since fibrin prevented inactivation of plasmin by α2AP, the presence of α2AP inactivated plasmin in the fluid phase.

Glu-plasminogen I and II: Their activation by urokinase and streptokinase in the presence of fibrin and fibrinogen

Two isozymes of a native form of human plasminogen (plg), Glu-plg I and II, were isolated, Glu-plg I or II was activated by urokinase (UK) or streptokinase (SK) in the presence of fibrinogen or fibrin. The activation of Glu-plg I was enhanced more than that of Glu-plg II in the presence of fibrin. Fibrin caused better activation of both Glu-plg I and II than fibrinogen When fibrinolysis or fibrinogenolysis was measured, fibrin was degraded faster than fibrinogen after the activation of Glu-plg I and II by UK. These results suggest that the activation of Glu-plg I was enhanced more than that of Glu-plg II in the presence of fibrin or to less extent fibrinogen

The enhanced activation of Glu-plasminogen by urokinase in the presence of fibrin or des a fibrin as measured by the release of Bβ peptide and FDP

Fresh plasma was incubated either with urokinase (UK) alone or the mixture of UK and human thrombin or Reptilase. In a purified system Glu-plasminogen (Glu-plg) was incubated with fibrinogen or fibrinogen plus thrombin in the presence of UK. At intervals, aprotinin was added to stop the reactions and the amounts of Bβ peptide and fibrin(ogen) degradation products (FDP, FgDP) were measured by radioimmunoassay and enzyme immunoassay, respectively. Results obtained by using plasma showed that fibrin was degraded faster than fibrinogen upon the addition of UK to the plasma or plasma clotted with thrombin. Bβ peptide was released faster from the clot than plasma. The clot formation caused by Reptilase (release of fibrinopeptide A) was accompanied by increase in the release of Bβ 1–42 from des A fibrin (fibrin without fibrinopeptide A). In a purified system, fibrin was degraded faster than fibrinogen upon the activation of Glu-plg by UK. These results may correlate well with the observation that Glu-plg was activated better by UK in the presence of fibrin than fibrinogen.